Author

Date of Degree

Document Type

Degree Name

Program

Advisor

Diego Loayza

Committee Members

David Foster

Hualin Zhong

Emily Bernstein

Anu Janakiraman

Subject Categories

Cancer Biology | Cell Biology

Keywords

Cancer, tumorigenesis, DNA damage response, ATR kinase

Abstract

Integrity of the human genome is essential for viability and proliferation of human cells. Intrinsic (endogenous replication stress) or extrinsic (UV, chemotherapy drugs) agents threaten the stability of the genome by generation of single stranded (ss) DNA or double stranded (ds) DNA breaks. The DNA damage response (DDR) pathways are conserved in evolution and constitute systems that perform the surveillance, signaling, and repair of the damage in the nucleus. Unchecked and accumulation of DNA damage can lead to deleterious effects such as replication fork collapse, chromosome fusion and breakage. The dysregulations of DNA damage response pathways are hallmarks of tumorigenesis. ATR (ataxia telangiectasia mutated and rad-3 related) is the protein kinase activated by ssDNA damage. ATR is essential for life and is recruited by ssDNA bound replication protein A (RPA). RPA, the major mammalian ssDNA binding complex with three different subunits: 70, 32, and 14, and is essential for replication, recombination and repair. The majority of ssDNA binding activity resides in the large subunit (RPA70), which directly binds to ssDNA though its central OB fold domains. It was established that LIM protein Ajuba inhibits unscheduled ATR response, and associates with RPA. However, it was unclear whether the association occurred directly, or through additional factors. I found that the Ajuba-RPA interaction is direct, through the RPA70 subunit, and likely occurs in the nucleus during S phase. Upon replication stress, Ajuba-RPA interaction was reduced and Ajuba was shuttled out of the nucleus during replication stress. In addition, I mapped the regions that likely mediate this direct contact to the first LIM domain of Ajuba and the ssDNA binding OB folds of RPA70. These findings revealed additional facets of ATR signaling regulation, and underscored the implications of LIM proteins in genome integrity and tumorigenesis.

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